Recent acceptance of the cholesterol 27-hydroxylation metabolic pathway as "mainstream" mandates the validation of a minimally invasive method that quantifies the rate of bile acid production and that is applicable to intact humans and experimental models of human disease. Because cholesterol 27-hydroxylase is expressed in many tissues that contribute to the normally circulating plasma pool of 27-hydroxycholesterol, we propose to validate the constant infusion technique for quantifying the rate of bile acid synthesis by administering tracer amounts of deuterated 27-hydroxycholesterol intravenously to intact NZW rabbits. As part of the validation procedure we will also determine bile acid production via the cholesterol 7alpha-hydroxylation metabolic pathway and compare the data obtained by the constant infusion method to that derived from the well-established isotope decay technique for total bile acid synthesis. After the validation studies, we will evaluate in NZW rabbits recent data implying that the cholesterol 27-hydroxylase pathway is specific for the metabolism of dietary cholesterol to bile acids. A group of male and female animals will alternate at 3 to 4 week intervals between a regular chow diet and that supplemented with cholesterol. Bile acid production via each metabolic pathway will be determined at the end of each period. Endogenous and deuterated hydroxysterols and bile acids will be analyzed by GLC-mass spectrometry using a simultaneous ion monitoring program after initial purification by thin-layer chromatography. Validation of this minimally invasive technique provides a novel approach for evaluating cholesterol metabolism in both neonates and adults and will provide greater understanding of the pathophysiology of diseases ranging from cholestasis to accelerated atherosclerosis.